JXB Advance Access originally published online on January 12, 2006
Journal of Experimental Botany 2006 57(3):655-664; doi:10.1093/jxb/erj055
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RESEARCH PAPER |
Water uptake by roots of Hordeum marinum: formation of a barrier to radial O2 loss does not affect root hydraulic conductivity
1School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley 6009, Western Australia
2CRC for Plant-Based Management of Dryland Salinity, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley 6009, Western Australia
3Lehrstuhl für Pflanzenoekologie, Universitaet Bayreuth, D-95440 Bayreuth, Germany
* To whom correspondence should be addressed. E-mail: tdcolmer{at}cyllene.uwa.edu.au
Received 5 May 2005; Accepted 11 November 2005
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Abstract |
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The adventitious roots of Hordeum marinum grown in stagnant deoxygenated solution contain a barrier to radial O2 loss (ROL) in basal zones, whereas roots of plants grown in aerated solution do not. The present experiments assessed whether induction of the barrier to ROL influences root hydraulic conductivity (Lpr). Wheat (Triticum aestivum) was also studied since, like H. marinum, this species forms aerenchyma in stagnant conditions, but does not form a barrier to ROL. Plants were grown in either aerated or stagnant, deoxygenated nutrient solution for 21–28 d. Root-sleeving O2 electrodes were used to assess patterns of ROL along adventitious roots, and a root-pressure probe and a pressure chamber to measure Lpr for individual adventitious roots and whole root systems, respectively. Lpr, measured under a hydrostatic pressure gradient, was 1.8-fold higher for individual roots, and 5.6-fold higher for whole roots systems, in T. aestivum than H. marinum. However, there was no difference in Lpr between the two species when measured under an osmotic driving force, when water moved from cell to cell rather than apoplastically. Root-zone O2 treatments during growth had no effect on Lpr for either species (measured in aerobic solution). It is concluded that induction of the barrier to ROL in H. marinum did not significantly affect the hydraulic conductivity of either individual adventitious roots or of the whole root system.
Key words: Adventitious roots, aerenchyma, exodermis, hydraulic conductivity, hypodermis, O2-deficiency, pressure chamber, radial O2 loss, root pressure probe, Triticum aestivum, wheat
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Introduction |
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The external cell layers in the sub-apical regions of adventitious roots of many wetland plants develop a very low permeability to O2, i.e. a barrier to restrict radial O2 loss (ROL) (Armstrong, 1979
; Armstrong et al., 2000; Colmer, 2003b). The barrier to ROL enhances longitudinal O2 diffusion within aerenchymatous roots towards the root tip, and thus root growth into anaerobic waterlogged soils (Armstrong, 1979; Jackson and Armstrong, 1999). A barrier to ROL is induced by growth in stagnant, deoxygenated conditions in a number of wild Hordeum species that inhabit wetland areas; for example, Hordeum marinum (Garthwaite et al., 2003). In contrast to these wild species, their cultivated, dryland relatives (barley, Hordeum vulgare, and wheat, Triticum aestivum) do not form a barrier to ROL (McDonald et al., 2001). Wheat, barley, and H. marinum all form adventitious roots containing aerenchyma, but wheat and barley are much less tolerant of waterlogging than H. marinum (McDonald et al., 2001).
In H. marinum grown in stagnant, deoxygenated nutrient solution, the apparent O2 diffusivity across the cell layers external to the root aerenchyma was 27-fold lower in the basal regions (which have very low rates of ROL) compared with near the root tip (a location with high rates of ROL) (AJ Garthwaite et al., unpublished data). The low O2 permeability near the base of the root was associated with secondary thickening, putatively lignin or suberin deposits, in the two outermost layers of the hypodermis (AJ Garthwaite et al., unpublished data). Oxygen micro-electrode studies of roots of Phragmites australis, which also forms a barrier to ROL, showed high resistance to O2 movement across the epidermal/hypodermal layers (Armstrong et al., 2000). Other structures that have been associated with a barrier to ROL include layers of sclerenchymatous fibres with thickened secondary walls (Oryza sativa; Clark and Harris, 1981), or tight, multi-layered hexagonal cell packing that eliminates gas spaces in the outer root tissues (numerous species, Justin and Armstrong, 1987; Armstrong et al., 1991).
Radial variation in the structure of root tissues can affect water uptake through roots, for a given ‘driving force’ (Steudle and Peterson, 1998; Lee et al., 2005; Schreiber et al., 2005). The effect on water uptake of changes in structure of the hypodermal layers in roots, albeit changes not associated with a barrier to ROL, have previously been evaluated for Agave desertii, Zea mays (maize), and Allium cepa (onion) (Melchior and Steudle, 1993). For example, development of an exodermis in Z. mays roots coincided with a 4-fold reduction in hydraulic conductivity (Zimmermann and Steudle, 1998). In roots of A. cepa, radial hydraulic conductivity decreased by a factor of five in the basal regions of the root where Casparian bands were present in the exodermis, and suberin lamellae had formed in the endodermis and exodermis (Melchior and Steudle, 1993). In Z. mays roots having an exodermis, there were significantly higher levels of aliphatic suberin and, to a lesser degree, lignin, in the basal half (Zimmermann et al., 2000). The presence of these compounds in the external cell layers has been related to a reduction in apoplastic water flow (Zimmermann et al., 2000). On the other hand, an increase in the amounts of suberin deposited in the exo- and endodermis of Z. mays and O. sativa did not necessarily reduce the resistance to radial water flow (Schreiber et al., 2005).
Adventitious roots of O. sativa constitutively form an exodermis (hypodermis with Casparian bands), and a layer of sclerenchymatous fibres with thickened secondary walls at the outer cortex (Clark and Harris, 1981). These hypodermal structures (in conjunction with a well-developed endodermis), were hypothesized to contribute to the ‘low’ root hydraulic conductivity measured for O. sativa (compared with Z. mays) (Miyamoto et al., 2001). However, by means of pressure-perfusion of solution through the large cortical air spaces in O. sativa adventitious roots, it was possible to measure the hydraulic conductivity of the cell layers external to the aerenchyma. Hydraulic conductivity of the external cell layers was 30-fold higher than the overall root hydraulic conductivity, indicating that the ‘apoplastic barriers’ in the external cell layers of O. sativa roots do not limit water uptake (Ranathunge et al., 2003). The barrier to ROL in adventitious roots of O. sativa is induced by growth in stagnant conditions (Colmer et al., 1998; Colmer, 2003a), however, a comparative analysis of the hydraulic conductivity for O. sativa roots with and without the barrier to ROL has not yet been performed, so it is not possible to draw conclusions on the effect of the barrier to ROL per se on water uptake by roots of O. sativa.
To determine whether or not induction of a barrier to ROL influences water uptake, the present study compared the hydraulic properties for individual adventitious roots, and whole root systems, of H. marinum (a species that forms a barrier to ROL) and T. aestivum (a species without a barrier to ROL) grown in aerated or stagnant, deoxygenated nutrient solution.
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Plant materials and culture
Hordeum marinum ssp. gussoneanum (accession H 819, Nordic Gene Bank, Alnarp, Sweden) and Triticum aestivum cv. ‘Crocus’ were used. H. marinum forms a barrier to ROL when grown in stagnant, deoxygenated nutrient solution, whereas T. aestivum does not (see Introduction).
Seeds were placed on plastic mesh floating on 0.1-strength aerated nutrient solution in darkness in a 20/15 °C (12 h day/night) controlled environment chamber (PAR 500 µmol m–2 s–1). The composition of the nutrient solution at full strength was (mol m–3): K+ 5.60, Ca2+ 1.50, Mg2+ 0.40, 
0.625, 
4.375, 
1.90, 
0.20, Na+ 0.20, 
0.10; and the micronutrients (mmol m–3): Cl– 50, B 25, Mn 2, Zn 2, Ni 1, Cu 0.5, Mo 0.5, Fe-Na-ethylenediaminetetra-acetic acid 50. The solution also contained 2.5 mol m–3 2-[N-morpholino]ethanesulphonic acid (MES) and the pH was adjusted to 6.5 with KOH (the final K+ concentration as above). All chemicals used were analytical grade.
Seedlings were exposed to light 4 d after imbibition and given 0.25-strength nutrient solution for an additional 4 d, before being transferred to pots of aerated, full-strength nutrient solution. Each 4.5 dm3 plastic pot held four seedlings. Plants were held in the lids using polystyrene foam holders. The pots and lids were covered with aluminium foil to ensure the roots were in darkness.
Root-zone O2 treatments
Eight replicate pots of each root-zone O2 treatmentxspecies combination were grown, with each complete replicate block germinated one week apart over a series of eight weeks to ensure a supply of plants of the correct age throughout the experimental period. Each pot contained four seedlings. Pots were arranged randomly within blocks. Seven days after the seedlings were transplanted into pots, root-zone O2 treatments were initiated (i.e. on 15-d-old plants). In the pots designated for the aerated treatment, the nutrient solution continued to be bubbled with air. For the remaining pots, designated for the stagnant treatment, the nutrient solution was replaced with de-oxygenated nutrient solution containing 0.1% (w/v) agar; the agar prevented convective movements in the solution (Wiengweera et al., 1997). Solutions in the aerated pots were also renewed at this time with aerated nutrient solution (without agar). All solutions were renewed every 7 d. Treatments were maintained for 21–28 d, with measurements taken during the final 7 d.
Radial O2 loss (ROL) from intact adventitious roots in an O2-free medium
ROL was measured on three replicates of each speciesxroot-zone O2 treatment combination to confirm the profiles of ROL were as previously found for these species (McDonald et al., 2001; Garthwaite et al., 2003). Plants were sealed just above the root–shoot junction into rubber lids fitted in 100x100x170 mm Perspex chambers filled with an O2-free solution containing 0.1% (w/v) agar and (mol m–3) Ca2+, 0.5 and K+, Cl– 5.0. The intact root system was immersed in O2-free solution while the shoot remained in air. A root-sleeving O2 electrode (height 5 mm; internal diameter 2.25 mm) (Armstrong and Wright, 1975; Armstrong, 1994) fitted with guides was placed around a selected adventitious root with a length in the range of 100–120 mm. The flux of O2 from the root to the electrode was measured with the centre of the electrode positioned at 80, 70, 60, 40, 20, and 5 mm behind the apex. The first measurements were taken at least 2 h after the plants were transferred into the system. All measurements were taken at 20 °C in a temperature-controlled room with photon-flux density at shoot height of 100 µmol m–2 s–1.
Measurements of ROL were also conducted to confirm that the barrier to ROL, induced in adventitious roots of H. marinum by growth in stagnant, deoxygenated nutrient solution, was still effective after the plant had been in aerated nutrient solution for 12 h. This enabled root pressure probe and pressure chamber experiments (described below) to be conducted in aerated nutrient solution.
Hydraulic conductivity of individual adventitious roots or whole root systems
The hydraulic conductivity was compared for individual adventitious roots and whole root systems of H. marinum and T. aestivum grown in aerated or stagnant nutrient solution. In all cases, hydraulic conductivity was measured with individual roots, or whole root systems, in aerobic conditions as this enabled it to be determined whether or not induction of the barrier to ROL impacts on root hydraulic conductivity, while avoiding possible direct effects of O2 deficiency on hydraulic conductivity during the measurements.
Using the root pressure probe to measure hydraulic conductivity of individual adventitious roots
Root pressure probe measurements were conducted as previously described (Steudle et al., 1987; Steudle and Frensch, 1996). In brief, excised roots with tips (100–140 mm) were connected to a root pressure probe using silicon seals to ensure root pressure was maintained without blocking xylem vessels. Roots attached to the probe were bathed in aerated nutrient solution with 20 mol m–3 glucose. Following attachment of roots, the system was left for at least 5 h or until stable root pressures (Pr, MPa) were established. Hydrostatic relaxations were performed by either increasing or decreasing the xylem pressure (using the probe). Osmotic relaxations were performed by suddenly increasing or decreasing the osmotic pressure of the external solution which was running through 0.25 m pipe (diameter=6 mm) with the fixed root protruding into it at one end. The resulting transient responses in pressure allowed hydraulic conductivity (Lpr) to be calculated from half-times of pressure relaxations ( 
) using the equation:
 | (1) |
Pr/Vs is the elastic coefficient of the measuring system, determined by inducing step changes in volume (VS) using the pressure probe and recording the resulting changes in root pressure (P); Ar is the surface area of the root, calculated by measuring the root length and average diameter. Measured root pressure relaxations usually showed a slower phase at the end of the relaxation, occupying about 20% of the entire relaxation. This has been attributed to a reversible concentration polarization of solutes at the endodermis (Steudle and Frensch, 1989; Ye and Steudle, 2005). This part of the curve was omitted in the analysis of relaxations.
In osmotic experiments, the external unstirred layer around the root may have contributed to the measured values of Lpr (‘gradient-dissipation effect’; Steudle and Frensch, 1989; Ye and Steudle, 2005). However, at flow rates along the root of usually between 0.3 and 0.5 m s–1, the effect should be negligible (see Results).
Osmotic experiments used additions of 25 mol m–3 NaCl to the bathing solution. The permeability coefficient for a given solute (Psr) is given by:
 | (2) |
is the half-time of solute exchange and Vx is the volume of functioning (mature) xylem, estimated by examining root cross-sections (data not shown); Ar as defined in equation 1.
Root reflection coefficients (
sr) were calculated from:
 | (3) |
denotes the change in osmotic pressure of the medium, ksr is the rate constant of permeation of a given solute (NaCl), and tmin is the time required to reach the minimum root pressure in the presence of the permeating solute ‘s’. As with the osmotic relaxations, measurements of permeability and reflection coefficients of roots can be influenced by effects of external unstirred layers. However, at the flow rates used in these experiments, 0.3–0.5 m s–1, these effects are likely to be small, as discussed in the Results section.
Hydraulic conductivity of whole root systems: measurement of root exudation in the presence of gradients in osmotic or hydrostatic pressure
Root exudation under an osmotic pressure gradient:
Tillers were cut 40–70 mm above the shoot base. All tillers except the main stem were sealed using clamps. Xylem sap exuding from the cut surface of the main stem was collected using a syringe and then weighed. Water uptake by the root was driven by the difference in osmotic pressure (
in MPa) between the medium and the xylem sap:
 | (4) |
sr denotes the root's reflection coefficient for the solutes in the external nutrient solution which was determined from experiments with the root pressure probe (described above). The osmotic pressure of the solution and exuded sap was measured using a freezing-point depression osmometer (Osmomat 030; Gonotec, Berlin, Germany).
Root exudation under a hydrostatic pressure gradient:
The root systems used to measure osmotic water flow were also used to measure hydraulic conductivity of the root system in the presence of hydrostatic pressure gradients. The root system was enclosed in a steel chamber where pneumatic pressure was applied to the root medium. Silicon seals and flexible rubber was placed around the stem to ensure a tight seal. The pressure in the chamber was raised in steps of 0.05 MPa and typically measurements were taken between 0.05 and 0.3 MPa above atmospheric. Exuded xylem sap was collected as described above, and the volume (V in m3) determined. The volume of exuded sap was plotted with time at a given applied pressure (Pgas in MPa). For the resulting linear relationship, the calculated slope was divided by the whole root surface area (m2, see below) to give the volume flow, JVr in m3 m–2 s–1. When plotting this against the applied pressure, the slope represented the hydraulic conductivity. The osmotic component was negligible for Pgas >0.05 MPa (Miyamoto et al., 2001). The technique resulted in an average value of the hydraulic resistance to water transport between all roots and the main stem when the tillers were clamped off. Root hydraulic conductivity (Lpr) was calculated from linear ranges of JVr (Pgas) curves.
Surface area of the whole root system:
The surface area of the whole (seminal and adventitious) root system for H. marinum and T. aestivum plants grown in aerated or stagnant nutrient solution was measured to enable calculations of whole root hydraulic conductivity (as described above). Root system surface area was determined using a video camera and image analysing software (Skye Instruments, Llandrindod Wells, UK). For better contrast, roots were stained with toluidine blue (0.03% w/v) prior to scanning. The surface area of the root system was calculated from projected areas of roots, assuming the roots to be cylindrical. The system was calibrated using metal wires of known lengths and diameters.
Statistics
ANOVA was performed on the various data sets to evaluate species and root-zone O2 treatment effects. Means were compared using the LSD (P=0.05), and are presented in the tables and figures accompanied by standard errors (GenStat, 6th edn, VSN International, www.vsn-intl.com).
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Results |
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Radial O2 loss (ROL) from intact adventitious roots in an O2-free medium
Figure 1 shows the induction of a barrier to ROL in the adventitious roots of stagnantly treated H. marinum. ROL near the base of the root was low (resistance to ROL high) and ROL rose sharply towards the root tip. By contrast, for roots previously in aerated solution, ROL was highest at the base of the root and it declined towards the root tip. Adventitious roots of T. aestivum grown in aerated solution had very low rates of ROL, which increased for roots in stagnant solution, with most ROL from basal regions; i.e. the absence of a barrier to ROL. These results confirmed that the experiments on hydraulic conductivity were performed on roots with, or without, a barrier to ROL.
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2.5 leaf stage in aerated solution and then grown in either aerated (open circles) or stagnant (filled circles), deoxygenated nutrient solution for a further 21–28 d. Measurements were taken at 20 °C, using roots 100–120 mm in length. Values are means ±SE (n=3).Hydraulic conductivity of individual adventitious roots
When measured using hydrostatic pressure gradients, the hydraulic conductivity (Lpr) of adventitious roots of T. aestivum was 1.8-fold higher than values for roots of H. marinum (P <0.05; Table 1). Growth in the stagnant nutrient solution did not affect hydrostatic Lpr of roots of either species (Table 1).
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Lpr, when measured using an osmotic pressure gradient, did not differ significantly between T. aestivum and H. marinum, nor did the root-zone O2 treatments affect Lpr, which ranged from 1.0x10–8 to 1.5x10–8 m s–1 MPa–1 in roots of both species from the two treatments (Table 1). The ratio of hydrostatic/osmotic estimates of Lpr was lower in H. marinum roots, being 5.1 and 4.0 in plants grown in aerated and stagnant nutrient solution, respectively, compared with 6.2 and 8.3, respectively, for roots of T. aestivum.
There was no marked difference in solute permeability (i.e. Psr of NaCl) between roots of T. aestivum and H. marinum, however, in plants grown in the stagnant nutrient solution, Psr was significantly higher in both species (P <0.05; Table 1). Psr ranged from 1.5–1.6x10–9 m s–1 in the aerated nutrient solution, compared with 2.3–2.5x10–9 m s–1 in the plants grown in stagnant solution. The reflection coefficient (sr) for individual adventitious roots for NaCl did not significantly differ between the two species, or root-zone O2 treatments, ranging from 0.21 to 0.30 (Table 1).
The steady-state root pressures (Pr) varied between 1.0–1.6x10–1 MPa (Table 1). T. aestivum had root pressures that were 20% higher, on average, than H. marinum (P <0.05; Table 1). Root pressures were between 18% and 40% higher in roots from plants grown in the stagnant solution (P <0.05; Table 1).
The half-time for hydrostatic pressure relaxations () ranged from 17 s to 24 s, while those for the osmotic pressure relaxations () ranged from 102 s to 152 s (Table 1). Since these parameters were used to calculate Lpr, statistical analysis was not performed on the data.
For these experiments the external medium was flowing around the roots at rates between 0.3 and 0.5 m s–1. This caused a vigorous stirring of the internal solution as discussed in an earlier paper (Ye and Steudle, 2005). Hence, the thickness of unstirred layers was estimated to be no bigger than around 50 µm. Assuming a slab geometry of the unstirred layer, this refers to a half-time of complete exchange of osmolytes of 
=0.5 s; calculated as: =0.281x(5x10–5)2/(1.5x10–9); where Ds of NaCl=1.5x10–9 m2 s–1 (Jost, 1960; Ye and Steudle, 2005). This was certainly not rate-limiting at half-times of water exchange of the root of 102–152 s during the osmotic experiments (Table 1).
It may be argued that the diffusion of solutes within the root apoplast, from the root surface to the exodermis and endodermis, and the diffusion in the stele (from the endodermis) to mature vessels, were contributing to the overall osmotic 
This may be true. However, model calculations of Steudle and Frensch (1989) for roots of Z. mays have shown that the effects are rather small (<10%). Moreover, the contributions of different internal pathways is equally interesting, depending on the type of experiments. If there are, for example, exchanges of water between the cell-to-cell and apoplastic path, this should affect the absolute value of Lpr, i.e. the apoplastic passage will be reflected in the overall measured parameter 
besides the cell-to-cell component (see Discussion). The same is true for the measured values of Psr and reflection coefficients. During the measurement of these latter parameters, effects of unstirred layers should have been even smaller because of the longer half-times of the exchange of solutes (for discussion of effects of unstirred layers, the reader is referred to Steudle and Frensch, 1989; Ye and Steudle, 2005).
Hydraulic conductivity of whole root systems: measurements of root exudation in the presence of an osmotic or a hydrostatic pressure gradient
Hydrostatic exudation (exudation in the presence of a hydrostatic pressure gradient) for the whole root system was significantly higher in T. aestivum than in H. marinum (P <0.05; Table 2). Hydrostatic conductivity of H. marinum was 0.8x10–8 m s–1 MPa–1 in both the aerated and stagnantly treated plants. In T. aestivum, hydrostatic exudation was 2.6x10–8 m s–1 MPa–1 in the plants raised in aerated solution, but was markedly higher at 6.4x10–8 m s–1 MPa–1 in those from the stagnant solution.
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Osmotic exudation (root exudation in the presence of an osmotic pressure gradient) for the whole root system did not differ significantly between T. aestivum and H. marinum, or for the plants from either root-zone O2 treatment; values of the osmotic water permeability ranged from 0.8–1.9x10–8 m s–1 MPa–1 (Table 2).
The ratio of hydrostatic/osmotic exudation for the whole root system was 1.0 for H. marinum grown in either aerated or stagnant nutrient solution. The same ratio was 2.4 for T. aestivum in the aerated solution, and it was 3.4 for plants in the stagnant solution.
Surface area of the whole root system
In the aerated nutrient solution, whole (seminal and adventitious) root surface area was 2.6-fold higher in T. aestivum compared with H. marinum (Table 2). In the stagnant nutrient solution, whole root surface area increased by 67% in H. marinum, whereas it decreased by 53% in T. aestivum (P <0.01; Table 2).
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This study demonstrates that formation of a barrier to radial O2 loss (ROL) in adventitious roots of H. marinum does not significantly affect root hydraulic conductivity. The barrier to ROL was induced in adventitious roots of H. marinum by growth in stagnant solution (Fig. 1); however, measurements of root hydraulic conductivity for both individual adventitious roots and whole root systems showed no difference between plants from aerated or stagnant treatments; i.e. with or without the barrier to ROL (Tables 1, 2). Thus, hypodermal structures putatively associated with the barrier to ROL in the roots of H. marinum (i.e. deposits of suberin or lignin in the hypodermis) (AJ Garthwaite et al., unpublished data) did not affect hydraulic conductivity. This result suggests that formation of a barrier to ROL may not necessarily impede other root functions, such as water uptake. Thus, although such trade-offs for root functioning had previously been hypothesized (reviewed by Colmer, 2003b
), the few data available do not support this notion, although studies of root functioning of a wider range of wetland species are needed to evaluate this hypothesis fully.
For H. marinum and T. aestivum, the hydraulic conductivity for individual adventitious roots was on average, 4.5- and 7.2-fold higher, respectively, when measured using a hydrostatic pressure gradient compared with an osmotic gradient. Since effects of unstirred layers could be excluded (see Results), this indicates a porous apoplastic bypass (Steudle and Peterson, 1998). Root pressure probe studies using Zea mays (maize) (Steudle et al., 1987; Steudle and Frensch, 1989) and Oryza sativa (rice) (Ranathunge et al., 2003) have given similar ratios between hydraulic conductivity measured using hydrostatic- or osmotically-induced water flows (Table 1; Steudle et al., 1987; Steudle and Frensch, 1989). This phenomenon has also been reported for other herbaceous plants (Table 1) and has been interpreted using the composite transport model (Steudle and Peterson, 1998); i.e. that in the absence of a hydrostatic pressure gradient, the apoplastic path is inefficient due to its very low reflection coefficient (). As such, water will flow predominantly via the protoplastic (cell-to-cell) path in response to an osmotic driving force.
That root hydraulic conductivity was not reduced by the presence of a putative apoplastic barrier (the barrier to ROL), might indicate that the cell-to-cell pathway is the dominant pathway of water uptake in H. marinum as already suggested for Hordeum distichon (syn. Hordeum vulgare) by Steudle and Jeschke (1983). However, work on O. sativa (Ranathunge et al., 2004) indicates that the apoplast is the dominant pathway for water uptake in the external cell layers, even in the presence of Casparian bands and suberin lamellae in the hypodermis/exodermis. Moreover, Schreiber et al. (2005) showed that, although the external cell layers of O. sativa roots contained significantly more suberin (per tissue surface area) than those of Z. mays, this did not contribute to the lower overall hydraulic conductivity of O. sativa roots compared with those of Z. mays (Ranathunge et al., 2003), leaving the authors to suggest that suberin deposits in root cell walls do not necessarily equate to reduced water and ion transport.
Further evidence of a dominating apoplastic pathway for water flow in the external cell layers of roots of O. sativa, was revealed (Ranathunge et al., 2004) by a comparison of diffusional and osmotic (bulk) H2O permeability. The diffusional H2O permeability of the external cell layers of O. sativa roots was 600–1400 times lower than the osmotic H2O permeability. The authors suggest that a high bulk versus diffusional H2O flow in roots of O. sativa could enable water uptake (via bulk H2O permeability), even when the external cell layers show low diffusional permeability to O2 (although O2 diffusion was not measured by Ranathunge et al., 2004). In the case of radial O2 diffusion across the outer part of the roots, consumption of O2 in respiration by the cells across the diffusion path would further diminish O2 flux from the exterior of the root (cf. Armstrong et al., 2000). Nevertheless, diffusional H2O permeabilities for O. sativa were 3-fold larger near the root tip than in the basal regions of the root (Ranathunge et al., 2004). However, since the plants used by Ranathunge et al. (2004) were not grown in stagnant conditions, a ‘tight’ barrier to ROL might not have been induced (Colmer et al., 1998; Colmer, 2003a) and actual differences in diffusional H2O permeability between the base and tip of the roots of O. sativa might be significantly larger when the barrier to ROL is fully induced. By analogy with O. sativa, water flow in roots of H. marinum could also occur via both the apoplastic and cell-to-cell pathways, despite the presence of putative apoplastic barriers.
The absolute values of individual root hydraulic conductivity of H. marinum and T. aestivum (present study) are comparable to those measured for O. sativa (Miyamoto et al., 2001; Ranathunge et al., 2003). However, Miyamoto et al. (2001) considered the value for O. sativa to be ‘low’ compared with roots of Z. mays, and this suggestion has been taken up by others (Ranathunge et al., 2003; Schreiber et al., 2005). When measured using a hydrostatic pressure gradient, the root hydraulic conductivity of O. sativa (Miyamoto et al., 2001; Ranathunge et al., 2003) was between 16–45% of values for Z. mays (Steudle et al., 1987, 1993; Steudle and Frensch, 1989) (Table 1). However, when compared with values of root hydraulic conductivity for other herbaceous species, including H. distichon (syn. H. vulgare), Phaseolus coccineus, and Allium cepa (Table 1), the values of hydraulic conductivity presented here for H. marinum and T. aestivum (and previous measurements of O. sativa) are not particularly low. Similarly, when root hydraulic conductivity was measured using an osmotic pressure gradient, the values for H. marinum and T. aestivum were also comparable to those of the species listed above (Table 1). Thus, root hydraulic conductivity of Z. mays might be considered to be at the higher end of this range for herbaceous species, and that for O. sativa does not appear to be abnormally low.
Whole root hydraulic conductivity of T. aestivum measured in the present study was 5.9-fold higher than in an earlier study (Gallardo et al., 1996). In the earlier study, hydraulic conductivity of the whole root system of 30–36-d-old T. aestivum growing in pots of soil was measured under a hydrostatic pressure gradient (Gallardo et al., 1996). The lower hydraulic conductivity of roots measured by Gallardo et al. (1996) compared with the present study presumably reflects, to a large degree, growth in soil compared with in hydroponics (cf. Z. mays, Zimmermann and Steudle, 1998).
The hydraulic conductivity measured in this study for individual roots of H. marinum and T. aestivum were generally higher than those measured for the whole root systems. In the case of H. marinum, the individual adventitious root hydraulic conductivities were 6.8- and 1.5-fold higher than those measured for the whole root systems, using hydrostatic or osmotic pressure gradients, respectively. Large differences between individual roots and whole root systems have not been observed in other species, i.e. O. sativa (values were essentially the same) (Miyamoto et al., 2001; Ranathunge et al., 2003) or Z. mays; individual roots 32% (hydrostatic gradients) to 58% (osmotic gradients) lower than whole systems (Steudle et al., 1987, 1993; Steudle and Frensch, 1989; Zimmermann and Steudle, 1998). This could be attributed to differences in root and shoot development between the plants of O. sativa and Z. mays used in the above studies, compared with the H. marinum and T. aestivum used in the present study. For example, the seedlings of Z. mays had small, simple root systems by comparison with the large seminal and adventitious root systems of H. marinum and T. aestivum. Furthermore, in O. sativa the adventitious root system dominates (only one seminal root is formed). Consequently, the individual root measurements made on O. sativa and Z. mays would better represent ‘whole’ root system measurements, than in the cases of H. marinum and T. aestivum. Moreover with H. marinum and T. aestivum having significantly more tillers (and adventitious roots originating from various tillers, resulting in flow pathways between vascular bundles of the tillers and main stem), the whole root system measurements will include any resistances to water movement between the tillers and main stem. Collectively, these differences in root and shoot development could contribute to the disparity in measured values of hydraulic conductivity of single adventitious roots, and whole root systems, for H. marinum and T. aestivum.
Roots of both H. marinum and T. aestivum grown in stagnant solution had higher values of Psr (NaCl) compared with plants grown in aerated solution. Psr (NaCl) in the aerated solution was 1.5–1.6x10–9 m s–1 for both species, whereas in the stagnant solution this increased to 2.3–2.5x10–9 m s–1. Waterlogging can reduce the capacity of roots to ‘exclude’ Na+ and Cl– (Barrett-Lennard, 2003). For example, in T. aestivum in 60 mol m–3 NaCl, 7 d of root-zone O2 deficiency imposed by flushing pots with N2, increased Na+ and Cl– concentrations in leaves by 240–330% (Barrett-Lennard, 2003). This is thought to occur via two processes (Barrett-Lennard, 2003), (i) loss of membrane integrity such that transpiration results in the uptake of ions by mass flow, and (ii) waterlogging having more specific effects on ion influx and efflux, mediated by deficits of energy (ATP). In the present experiments, Psr (NaCl) was measured in aerated solution, so the higher values for roots previously in stagnant solution suggests that membrane integrity of at least a portion of the root might have been compromised (e.g. cells most distant from the source of O2, such as the stele and root tips; Colmer and Greenway, 2005), and this did not recover within the first few hours following return to aeration. Furthermore, re-aeration following O2-deprivation can result in damage to membranes if reactive oxygen species (ROS) accumulate (Blokhina et al., 2003).
In the present study, plants were grown in stagnant, deoxygenated nutrient solution for up to 28 d prior to measurement of hydraulic conductivity in aerobic conditions. This enabled it to be determined whether or not induction of the barrier to ROL impacts on root hydraulic conductivity, while avoiding the possible direct influence of O2 deficiency on water uptake by roots. Short-term hypoxia, or anoxia, cause declines in root hydraulic conductivity, as shown for Z. mays (Birner and Steudle, 1993; Gibbs et al., 1998), Musa ssp. (banana) (Aguilar et al., 2003) and tomato (Jackson et al., 1996), although the effects may be transient (Jackson et al., 1996; Gibbs et al., 1998), indicating that the long-term effects of flooding on water uptake (see review by Kozlowski, 1984) may be an indirect, rather than a direct, effect of O2 deficiency (Gibbs et al., 1998). However, results presented here indicate, at least for H. marinum, that the formation of a barrier to ROL in the basal regions of adventitious roots, induced by growth in stagnant conditions, does not significantly affect root hydraulic conductivity.
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AJG thanks colleagues at the Lehrstuhl Pflanzenoekologie, Universitaet Bayreuth, Bayreuth, for technical assistance and kind support during a research visit in 2003. The Grains Research and Development Corporation provided a student scholarship for AJG.
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